First report of tobacco anthracnose caused by Colletotrichum nymphaeae in China.

Abstract

Tobacco (Nicotiana tabacum L.) is one of the most widely cultivated economic crops in approximately 120 countries (Peedin 2011). In July 2020 and 2021, typical symptoms of tobacco anthracnose were widely found in the flue-cured tobacco-planted areas of Wufeng, Xuan'en, and Xianfeng, Hubei Province, China. The disease incidence reached up to 60% in some fields at that time, with estimated 10,000 ha of the cultivated area affected. On tobacco leaves, lesions were initially water soaked and yellow green, and these enlarged to produce dark-brown necrosis which became cracked after drying, extending until the leaves withered. After surface-sterilization with 75% ethanol for 45 s and 5% sodium hypochlorite for 60 s, diseased leaf tissues were washed with sterilized water for 60 s three times and then cultured on potato dextrose agar (PDA) plates for seven days at 25°C in the dark. Isolates of Colletotrichum sp. were consistently recovered with isolation rate of 71%, and the five isolates BB005ES1, BB005ES2, BB005ES3, BB005ES4 and BB005ES5 were used to further evaluate characteristics of the pathogen. On PDA medium for seven days, the aerial hyphae of cultures were dense and blanket-like. The aerial surface of the colony was dark gray to white, and the center of the basal surface of the colony was orange-red. Conidia were transparent, aseptate, smooth-walled, straight, cylindrical with one end obtuse and the other end funnel-shaped, and the size was 11.8-12.0 μm×2.7-2.9 μm (n=100). Appressoria were single, smooth, black, oval or irregular shapes with size of 4.6-4.9 μm×8.5-8.7 μm (n=100). The most typical feature of Colletotrichum acutatum species complex is the shape of conidia which have at least one acute end (Damm et al., 2012). Thus, the five strains were identified as part of the Acutatum complex. The sequences of ACT, TUB2, CHS-1, GAPDH and ITS were then amplified from the five strains (Damm et al., 2012), and all the five strains had the similar sequence for each gene (Accession numbers in GeneBank: ON637946, ON637947, ON637945, ON637948 and ON394623). The combined sequences ACT-TUB2-CHS-1-GAPDH-ITS of the five strains were used for constructing multigene phylogenetic tree using Maximum Parsimony method (Prihastuti et al. 2009), and C. gloeosporioides (IMI356878) was selected as an outgroup. The five strains were found to be closely related to the type strains of C. nymphaeae. Hence, the five isolated strains were identified as C. nymphaeae. Pathogenicity of the five strains was determined by placing seven-day-old fungal plugs on attached leaves of 20-day-old tobacco plants in lab. After inoculation, plants were incubated in a 28°C and 95% RH incubator in the dark for five days. The five strains caused the typical dark brown lesions on all inoculated tobacco leaves, whereas no disease symptoms were found on the healthy tobacco leaves for agar-plug inoculation controls. Koch's postulates were fulfilled by re-isolating C. nymphaeae from diseased leaves. Previously, only C. fructicola, C. nicotiance, C. orbiculare and C. cliviicola were documented as causal agents of tobacco anthracnose (Wang et al. 2016；Wang et al. 2022). To our knowledge, this is the first report of C. nymphaeae causing tobacco anthracnose worldwide.